CN104906117A - Application of 1,6-fructose diphosphate in preparation of medicine for preventing and treating enveloped virus infection and virus inactivating agents - Google Patents
Application of 1,6-fructose diphosphate in preparation of medicine for preventing and treating enveloped virus infection and virus inactivating agents Download PDFInfo
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Abstract
The invention discloses an application of 1,6-fructose diphosphate and salt formed by the1,6-fructose diphosphate in the preparation of medicine for preventing and treating human enveloped virus infection and virus inactivating agents. The copying level of a variety of common human viruses pretreated by 1,6-fructose diphosphate sodium salt on cells is detected, and the virus killing activity of the 1,6-fructose diphosphate sodium salt on the various human viruses is evaluated. Results show that the 1,6-fructose diphosphate sodium salt has dose-dependent virus killing activity on enveloped viruses. Experiments prove that the virus killing function of the 1,6-fructose diphosphate sodium salt depends on temperature. The 1,6-fructose diphosphate is a glycolysis mesostate, and meanwhile the function of adjusting metabolism is achieved. The compound can inactivate a variety of enveloped viruses, and a broad prospect of developing the compound into the medicine for preventing and treating human enveloped virus infection and the virus inactivating agents is achieved.
Description
Technical field
The invention belongs to medical art, the application of salt in the medicine and external virus inactivating agent of preparation control mankind infection of coated virus more specifically relating to a kind of fructose 1,6-diphosphate and formed.
Background technology
One deck is had to derive from the cyst membrane of host cell outside the nucleocapsid of enveloped virus.Cyst membrane is one deck phospholipid bilayer tunic be wrapped in outside stromatin, and this tunic derives from the cell membrane of host, it is also inlayed the envelope protein of virus-specific.The dengue virus (DENV) of flaviviridae and Japanese B encephalitis virus (JEV), the vesicular stomatitis virus (VSV) of Rhabdoviridae and the respiratory syncytial virus (RSV) of Paramyxoviridae are tunicary virus, are all the important pathogen bodies of common threat human health.
Dengue virus is widely current in global tropical and semi-tropical more than 60 countries and regions, and infected per year over 100000000 people, the people of more than 2,500,000,000 is on the hazard.Dengue fever (dengue fever) is that dengue virus causes, her mosquito-borne a kind of acute infectious disease.Clinical symptoms is that onset is hurried, high heat, whole-body muscle, bone marrow and arthralgia, and extremely tired, part is suffered from can erythra, bleeding tendency and lymphadenectasis.Japanese B encephalitis (encephalitis b) is the acute infectious diseases common to human beings and animals caused by Japanese B encephalitis virus, clinically with symptom and signs such as high heat, disturbance of consciousness, tic, respiratory failure and meningeal irritation signs for feature, case fatality rate is about 25%.Dengue fever and encephalitis b, by mosquito media transmission, are popular in whole Asia, mainly at the east Asia torrid zone, subtropical zone and temperate countries.Encephalitis b mainly betides summer and autumn, and child easily suffers from, and sickness rate accounts for more than 80%, mostly is less than 10 years old child.For encephalitis b virus, though have vaccine at present, not special antiviral therapy.Encephalitis b virus infection remains one of human health and threatens greatly.The spread speed of influenza virus is fast, infectiousness is strong and local can be caused in the short time to break out the even whole world is very popular, from finding the emphasis always being global concern so far.In 20th century, IAV causes altogether and is very popular for three times.Wherein the large influenza of Spain of 1918-1920 outburst has the greatest impact, and causes the whole world 5,000 ten thousand people dead.1997, high pathogenic avian influenza appeared at Hong Kong, because the infection that the destructive outburst of poultry is dead and the mankind are dispersed in, and simultaneously adjoint very high mortality rate, the mankind are absorbed in great fear.Although this two classes pathogen is completely different in the source of infection, route of transmission and pathogenesis etc., human health is all made to be on the hazard and to bring economic loss.
At present, the disease caused for these enveloped viruses clinically mainly adopts interferon and ribavirin to carry out antiviral therapy.But medicine exists certain side effect, and relevant disease can not be cured completely.In addition, the life-time service of these medicines causes virus to create many medicament-resistant mutation strains.Therefore, be badly in need of development of new safely and effectively medicine prevent or inactivation of viruses infect.
Fructose 1,6-diphosphate is the important intermediate product of glycolysis metabolism approach, can play regulating action again simultaneously, be regarded as a kind of metabolic modifiers and energy-rich phosphate substrate to the metabolism of saccharide, lipid.At clinicing aspect, fructose 1,6-diphosphate is widely used, and can be used for myocarditis, angina pectoris, myocardial infarction, shock, the environment of ischemic hypoxia and the auxiliary treatment of hepatitis.But fructose 1,6-diphosphate remains blank out in the research of field of virology.Fructose 1,6-diphosphate structure is as follows:
Summary of the invention
The object of the invention is to make up the deficiencies in the prior art, there are provided a kind of 1,6-fructose diphosphate and the application in the medicine and external virus inactivating agent of preparation control mankind infection of coated virus of the salt that formed thereof, thus provide measure safely and effectively for the control of relevant disease caused by mankind's infection of coated virus clinically.Fructose 1,6-diphosphate and the salt that formed thereof can the infection of deactivation enveloped virus effectively within the scope of avirulence, can be developed as the medicine of the relevant disease that control enveloped virus causes and external virus inactivating agent further, be with a wide range of applications.
In order to realize above-mentioned object, the technical solution used in the present invention is:
A kind of fructose 1,6-diphosphate and the application of salt in the medicine and virus inactivating agent of preparation control mankind infection of coated virus formed thereof.
Preferably, described enveloped virus is Japanese B encephalitis virus (JEV), dengue virus (DENV), respiratory syncytial virus (RSV) and influenza A virus (IAV) each hypotype.
Comprise the following steps:
1. fructose 1,6-diphosphate sodium salt kills enveloped virus Activity determination, the steps include:
a. fructose 1,6-diphosphate sodium salt is to mdck cell, VERO cell, BHhK-21 cell and Hep-2 cytotoxicity experiment:
Mdck cell, VERO cell, BHhK-21 cell and Hep-2 cell are by 8 × 10
3individual cells/well is inoculated in 96 porocyte culture plates, for subsequent use after cell attachment.With culture medium by fructose 1,6-diphosphate sodium salt from 20mg/mL, 2 times of gradients are diluted to 9 concentration successively, the culture medium of drug containing are directly added in adherent various cells, often organize repetition three holes, be placed in 37 DEG C, 5% CO
2cultivate in incubator.The cell being added with medicine is placed in 37 DEG C, 5% CO
2cultivate in incubator after 48 ~ 96 hours, adopt Alamarblue activity detection kit to detect the survival rate of cell.
Result display fructose 1,6-diphosphate sodium salt is very low to the toxicity of these cells, half toxic concentration (CC
50) at 2000 more than μ g/mL, safety is fine.
Fructose 1,6-diphosphate sodium salt detects the viricidal activity of enveloped virus:
(1) influenza virus liquid (6.5 × 10
5tCID
50/ mL) with 1 of 3 times of gradient dilutions, 6-fructose diphosphate sodium (final concentration is respectively 1000 μ g/mL, 333 μ g/mL, 111 μ g/mL, 37 μ g/mL, 12.3 μ g/mL, 4.1 μ g/mL, 1.37 μ g/mL) 37 DEG C of preincubates 4 hours, then by mixture diluted 100 times of postoperative infection mdck cells; Matched group be then viral stock and each medicine respectively 37 DEG C of preincubates 4 hours, mdck cells are infected in each dilution 50 times afterwards mixing.Virus infected cell removed supernatant after 1 hour, washed twice with PBS, removed the virus and residual medicine of not adsorbing.Then be changed to fresh culture and continue cultivation after 24 hours at 37 DEG C, collect supernatant and measure neuraminidase (NA) activity, calculate suppression ratio.
(2) Japanese B encephalitis virus JEV-Luc liquid (8 × 10
6and dengue virus DENV-Luc liquid (4 × 10 pfu/mL)
7pfu/mL) respectively with 1 of 3 times of gradient dilutions, 6-fructose diphosphate sodium (final concentration is respectively 1000 μ g/mL, 333 μ g/mL, 111 μ g/mL, 37 μ g/mL, 12.3 μ g/mL, 4.1 μ g/mL, 1.37 μ g/mL) was 37 DEG C of preincubates 4 hours, again by virus and medicament mixed diluted sample 100 times of postoperative infection BHK-21 cells, cultivate 1 hour for 37 DEG C.Equally, cell PBS washes twice, after removing the virus and residual medicine of not adsorbing, is changed to fresh culture and continues cultivation 48 hours, detects cell fluorescence element enzymatic activity, calculates suppression ratio.
(3) blister stomatitis virus VSV-GFP virus liquid (4 × 10
7pfu/mL) with the fructose 1,6-diphosphate sodium salt (final concentration is respectively 1000 μ g/mL, 333 μ g/mL, 111 μ g/mL, 37 μ g/mL, 12.3 μ g/mL, 4.1 μ g/mL, 1.37 μ g/mL) of 3 times of gradient dilutions 37 DEG C of preincubates 4 hours.Virus and medicament mixed liquid are diluted 100 times of postoperative infection BhK-21 cells, cultivate after 1 hour for 37 DEG C and remove supernatant.Cell PBS washes twice, removes the virus and residual medicine of not adsorbing, is then changed to fresh culture.Continue cultivation after 12 hours, by high intension image checking and the fluorescence intensity of quantitative analysis green fluorescent protein, calculate suppression ratio.
(4) adenovirus hominis (ADV), enterovirns type 71 (EV71-GFP) respectively with 1 of 3 times of gradient dilutions, 6-fructose diphosphate sodium (final concentration is respectively 1000 μ g/mL, 333 μ g/mL, 111 μ g/mL, 37 μ g/mL, 12.3 μ g/mL, 4.1 μ g/mL, 1.37 μ g/mL) after 4 hours, with MOI=1 and MOI=0.2 infects VERO cell after diluting 100 times at 37 DEG C of preincubates.Cultivate after 1 hour for 37 DEG C and remove supernatant, wash twice with PBS, remove the virus and residual medicine of not adsorbing.Then be changed to fresh culture and continue cultivation after 48 hours, surveyed and quantitative analysis cell pathological changes (cytopathic effect, CPE) or the fluorescence intensity of green fluorescent protein by high intension inspection imaging system, calculate suppression ratio.
(5) respiratory syncytial virus RSV and diluted 1,6-fructose diphosphate sodium (final concentration is respectively 1000 μ g/mL, 333 μ g/mL, 111 μ g/mL, 37 μ g/mL, 12.3 μ g/mL, 4.1 μ g/mL, 1.37 μ g/mL) at 37 DEG C of preincubates after 3 hours, with 100TCID after diluting 100 times
50infect Hep-2 cell.Cultivate after 1 hour for 33 DEG C and remove supernatant, wash twice with PBS, remove the virus and residual medicine of not adsorbing.Then be changed to fresh culture and continue cultivation after 96 hours at 33 DEG C, active by CellTiter-Glo kit detection cell, calculate the suppression ratio of cytoprotective rate and drug on viral.
Result display fructose 1,6-diphosphate sodium salt has dose-dependent viricidal activity to the enveloped virus such as JEV, DENV, RSV and influenza virus.But fructose 1,6-diphosphate sodium salt does not but show viricidal activity to nonenveloped virus ADV and EV71.Illustrate that fructose 1,6-diphosphate sodium salt has the effect of killing the virus of wide spectrum to enveloped virus.
2. the influenza activity that kills of fructose 1,6-diphosphate sodium salt temperature-independent detects, and the steps include:
Influenza A virus [A/PuertoRico/8/34 (H1N1)] liquid (6.5 × 10
5tCID
50/ mL) with 1 of 3 times of gradient dilutions, 6-fructose diphosphate sodium (final concentration is respectively 1000 μ g/mL, 333 μ g/mL, 111 μ g/mL, 37 μ g/mL, 12.3 μ g/mL) hatches 4 hours 4 DEG C, 25 DEG C, 37 DEG C and 42 DEG C respectively, then by virus mixture diluted 100 times of postoperative infection mdck cells with medicine.Virus infected cell removed supernatant after 1 hour, washed twice with PBS, removed the virus and residual medicine of not adsorbing.Then be changed to fresh culture and continue cultivation after 24 hours at 37 DEG C, collect supernatant and measure NA activity, calculate suppression ratio.
Result shows that the effect of killing the virus of fructose 1,6-diphosphate sodium salt infected by influenza is temperature-independent.Fructose 1,6-diphosphate sodium salt is better than the inactivating efficacy of 4 DEG C and 25 DEG C at 37 DEG C and 42 DEG C of inactivating influenza virus effects.
The present invention compared with prior art, has the following advantages and effect:
1. clinically, fructose 1,6-diphosphate sodium salt is widely used in the auxiliary treatment of myocarditis, angina pectoris, myocardial infarction, ischemic hypoxia and hepatitis, and is the intermediate product of human body carbohydrate metabolism approach, little to the toxic and side effects of human body.
2. fructose 1,6-diphosphate sodium salt is under avirulent condition, can the infection of deactivation JEV, DENV, RSV and each hypotype of influenza virus effectively, can be made into the antiviral drugs of wide spectrum.
3. utilize modern common drug preparation means, fructose 1,6-diphosphate sodium salt can be made the pharmaceutically acceptable dosage forms of any one such as tablet, capsule, granule, oral liquid, slow releasing preparation, controlled release preparation, nanometer formulation or injection as active component.
4. fructose 1,6-diphosphate sodium salt has the function of inactivation of viruses, can be prepared into external virus inactivating agent.
Accompanying drawing explanation
Fig. 1 is fructose 1,6-diphosphate chemical structural formula.
Fig. 2 be fructose 1,6-diphosphate sodium salt temperature-independent kill influenza activity detect schematic diagram.
Detailed description of the invention
In order to understand content of the present invention better, below in conjunction with specific implementation method, content of the present invention is described further, but protection content of the present invention is not limited to following examples.
Embodiment 1:1,6-fructose diphosphate sodium detects the viricidal activity of each subtype influenza virus.
1. experiment material
1.1 cells, virus
Madin-Darby canine kidney(cell line) (MDCK) MDCK(ATCC CCL-34) preserved by this laboratory.Influenza A H1N1 hypotype (A/PuertoRico/8/1934 and A/human/Hubei/1/2009), H3N2 hypotype (A/ Human/Hubei/3/2005), H6N6 hypotype (A/Duck/Hubei/5/2010), H7N8 hypotype (A/Duck/Hubei/ 216/ 1983) and H1N1 hypotype amantadine persister (A/Human/WSN/33 (S31N)) are this laboratory and preserve, and increased and obtain by Embryo Gallus domesticus.Instar chicken embryo was purchased from Beijing Cimmeria Wei Tong laboratory animal Technology Co., Ltd. on 10th.
1.2 reagent
DMEM culture medium is purchased from GIBCO company; Alamarblue activity detection kit is purchased from Promega company; Neuraminidase substrate MUNANA is purchased from Sigma-Aldrich.Fructose 1,6-diphosphate sodium salt is purchased from Aladdin.
1.3 experimental apparatus
Multiple labeling microtiter plate reader (PerkinElmer) is Bole's Products; Cell culture incubator is Thermo Products.
2. experimental technique and result
2.1 fructose 1,6-diphosphate sodium salts detect the cytotoxicity of mdck cell.
(1) by mdck cell according to 8 × 10
3individual cells/well is inoculated in 96 porocyte culture plates, adopts containing 10% hyclone (FBS), the penicillin of 100U/mL and the DMEM culture medium of streptomycin.Cell, at 37 DEG C, is cultivated in 5% CO2 humidified incubator.
(2) with culture medium by fructose 1,6-diphosphate sodium salt from 20mg/ml, 2 times of gradients are diluted to 9 concentration successively, the culture medium of drug containing are directly added in adherent mdck cell, often organize repetition three holes, be placed in 37 DEG C, 5% CO
2cultivate in incubator.
After (3) 48 hours, utilize the cytotoxicity of Alamarblue activity detection kit detection of drugs.Read plate instrument (Envison 2102 Multilabel Reader) with PerkinElmer multiple labeling and read fluorescence signal.
(4) each group data calculate using not drug containing matched group as criterion group, computing formula=medicine group/do not add medicine * 100.Result goes out meansigma methods and standard deviation by GraphPad Prism 5 computed in software.
Utilize the result of calculation in step (4), by software statistics, statistical result is in table 1.Result display fructose 1,6-diphosphate sodium salt is to the Cytotoxic CC of mdck cell
50be 2800 μ g/mL.
2.2 fructose 1,6-diphosphate sodium salts are to the detection of each subtype influenza virus inactivating efficacy.
(1) influenza A H1N1 hypotype (A/PuertoRico/8/1934 and A/Human/Hubei/1/2009), H3N2 hypotype (A/ Human/Hubei/3/2005), H6N6 hypotype (A/Duck/Hubei/5/2010), H7N8 hypotype (A/Duck/Hubei/ 216/ 1983) and H1N1 hypotype amantadine persister (A/Human/WSN/33 (S31N)) viral stock respectively with 1 of 3 times of gradient dilutions, (final concentration is respectively 1000 μ g/mL to 6-fructose diphosphate sodium, 333 μ g/mL, 111 μ g/mL, 37 μ g/mL, 12.3 μ g/mL, 4.1 μ g/mL, 1.37 μ g/mL) 37 DEG C of preincubates 4 hours.Then by virus mixture diluted 100 times of postoperative infection mdck cells with medicine; Matched group be then viral stock and each acute drug respectively 37 DEG C of preincubates 4 hours, each dilution 50 times of rear mixings infect mdck cell immediately.
(2) virus infected cell removed supernatant after 1 hour, washed the virus and residual medicine removing for twice and do not adsorb with PBS, was then changed to fresh culture and continued to cultivate.
(3) cultivate after 24 hours, collect supernatant and measure NA(neuraminidase) active, calculate suppression ratio.
The each hypotype of result display fructose 1,6-diphosphate sodium salt energy dose-dependant ground inactivating influenza virus, by software statistics, the IC of each hypotype of fructose 1,6-diphosphate infected by influenza
50be 45 ~ 130 μ g/mL, the results are shown in Table 1.
The viricidal activity of embodiment 2:1,6-fructose diphosphate sodium to dengue virus and Japanese B encephalitis virus detects.
1. experiment material
1.1 cells and virus
Hamster kidney cell (BHK-21) is preserved for this laboratory.
Japanese B encephalitis virus (JEV-Luc) and dengue virus (DENV-Luc): for the Japanese B encephalitis virus of Rellina luciferase reporter gene and dengue virus, presented by Chinese Academy of Sciences Wuhan institute of viruses Zhang Bo researcher.
1.2 reagent
DMEM culture medium and FBS are purchased from GIBCO company; Alamarblue activity detection kit is purchased from Promega company;
renillaluciferase Assay Reagent box is purchased from Promega company.
1.3 experimental apparatus
Multiple labeling microtiter plate reader (PerkinElmer); High intension cytoanalyze (PerkinElmer); Megafuge 1.0R type refrigerated centrifuger and cell culture incubator are Thermo Products.
2. experimental technique and result
2.1 fructose 1,6-diphosphate sodium salts detect the cytotoxicity of BHK-21 cell.
(1) by BHK-21 cell according to 8 × 10
3individual cells/well is inoculated in 96 porocyte culture plates, uses the DMEM culture medium of the penicillin and streptomycin containing 10%FBS, 100U/mL at 37 DEG C, cultivates in the humidified incubator of 5% CO2.
(2) with culture medium by fructose 1,6-diphosphate sodium salt from 20mg/ml, 2 times of gradients are diluted to 9 concentration successively, the culture medium of drug containing are directly added in adherent BHK-21 cell, often organize repetition three holes, be placed in 37 DEG C, 5% CO
2cultivate in incubator.
After (3) 48 hours, utilize the cytotoxicity of Alamarblue activity detection kit detection of drugs.Read plate instrument (Envison 2102 Multilabel Reader) with PerkinElmer multiple labeling and read fluorescence signal.
(4) each group data calculate using not drug containing matched group as criterion group, computing formula=medicine group/do not add medicine * 100.Result goes out meansigma methods and standard deviation by GraphPad Prism 5 computed in software.
Result of calculation in step (4), is added up by computed in software, the results are shown in Table 1.Result display fructose 1,6-diphosphate sodium salt is to the Cytotoxic CC of BHK-21 cell
50be 7090 μ g/mL.
The viricidal activity of 2.2 fructose 1,6-diphosphate sodium salts to dengue virus and Japanese B encephalitis virus detects
(1) JEV-Luc(8 × 10
6pfu/ml) with DENV-Luc(4 × 10
6pfu/ml) respectively with 1 of 3 times of gradient dilutions, 6-fructose diphosphate sodium (final concentration is respectively 1000 μ g/mL, 333 μ g/mL, 111 μ g/mL, 37 μ g/mL, 12.3 μ g/mL, 4.1 μ g/mL, 1.37 μ g/mL) was 37 DEG C of preincubates 4 hours, again by virus and medicament mixed diluted sample 100 times, postoperative infection BHK-21 cell; Matched group be then viral stock and each acute drug respectively 37 DEG C of preincubates 4 hours, mix after each dilution 50 times and infect BHK-21 cell immediately.
(2) virus infected cell removed supernatant after 1 hour, washed twice with PBS, removed the virus and residual medicine of not adsorbing, and was then changed to fresh culture and continued to cultivate.
(3) after cultivating 48 hours at 37 DEG C, measure intracellular luciferase active, calculate suppression ratio.Cell PBS to be measured washes 2 times, then adds lysate by the method for Renilla Luciferase Assay Reagent box description and makes the abundant cracking of cell, then lysate sample added in the substrate diluted, luciferase detector detects.Computing formula is: suppression ratio (%)=100-(sample well-blank)/(enzyme contrast-blank alive) * 100%.
Experimental result display fructose 1,6-diphosphate sodium salt energy dose-dependant ground deactivation Japanese B encephalitis virus and dengue virus.Fructose 1,6-diphosphate sodium salt is to the IC of Japanese B encephalitis virus and dengue virus
50be respectively 112 and 6.8 μ g/mL, the results are shown in Table 1.
Embodiment 3:1,6-fructose diphosphate sodium detects the viricidal activity of vesicular stomatitis virus (VSV).
1. experiment material
1.1 cells, virus
Hamster kidney cell (BHK-21) is preserved for this laboratory.
VSV-GFP: be the vesicular stomatitis virus with GFP reporter gene, presented by Wuhan University professor Chen Mingzhou.
1.2 reagent
DMEM culture medium and FBS are purchased from GIBCO company; Alamarblue activity detection kit is purchased from Promega company.
1.3 experimental apparatus
High intension cytoanalyze (PerkinElmer); Zeiss senior inverted microscope Axio observer A1 (Carl Zeiss).
2. experimental technique and result
2.1 fructose 1,6-diphosphate sodium salts detect (see embodiment 2) the cytotoxicity of BHK-21 cell.
2.2 fructose 1,6-diphosphate sodium salts kill vesicular stomatitis virus Activity determination.
(1) VSV-GFP viral stock (4 × 10
7pfu/mL) with 1 of 3 times of gradient dilutions, 6-fructose diphosphate sodium (final concentration is respectively 1000 μ g/mL, 333 μ g/mL, 111 μ g/mL, 37 μ g/mL, 12.3 μ g/mL, 4.1 μ g/mL, 1.37 μ g/mL) 37 DEG C of preincubates 4 hours, then by virus and medicament mixed diluted sample 100 times of postoperative infection BhK-21 cells; Matched group be then viral stock and each acute drug respectively 37 DEG C of preincubates 4 hours, mix after each dilution 50 times and infect BHK-21 cell immediately.
(2) virus infected cell removed supernatant after 1 hour, washed twice with PBS, removed the virus and residual medicine of not adsorbing, and was then changed to fresh culture and continued to cultivate.
(3) cultivate after 12 hours, detected and the fluorescence intensity of quantitative analysis green fluorescent protein by high intension, calculate suppression ratio.Suppression ratio (%)=(viral hole fluorescence intensity-sample well fluorescence intensity)/viral hole fluorescence intensity * 100%.
Experimental result display fructose 1,6-diphosphate sodium salt energy dose-dependant ground deactivation vesicular stomatitis virus, fructose 1,6-diphosphate sodium salt is to the IC of vesicular stomatitis virus
50be 12 μ g/mL, the results are shown in Table 1.
The viricidal activity of embodiment 5:1,6-fructose diphosphate sodium to adenovirus ADV and enterovirus EV 71 detects.
1. experiment material
1.1 cells, virus
VERO cell is the preservation of this laboratory.
Adenovirus (Adenovirus) is by Wuhan Virology Institute,Chinan academy of Sciences, and microbial bacteria seed culture of viruses resource is presented with application center.EV71-GFP virus: with EV71-GFP plasmid as template, after linearisation, in vitro transcription obtains full-length genome RNA, then obtain possessing infective virion with amplification after liposome transfection VERO cell.
1.2 reagent
DMEM culture medium and FBS are purchased from GIBCO company; Alamarblue activity detection kit is purchased from Promega company.
1.3 experimental apparatus
High intension cytoanalyze (PerkinElmer); Zeiss senior inverted microscope Axio observer A1 (Carl Zeiss).
2. experimental technique and result
2.1 fructose 1,6-diphosphate sodium salts detect the cytotoxicity of VERO cell.
(1) by VERO cell according to 8 × 10
3individual cells/well is inoculated in 96 porocyte culture plates, uses the DMEM culture medium of the penicillin and streptomycin containing 10%FBS, 100U/mL at 37 DEG C, 5% CO
2humidified incubator in cultivate.
(2) with culture medium by fructose 1,6-diphosphate sodium salt from 20mg/ml, 2 times of gradients are diluted to 9 concentration successively, the culture medium of drug containing are directly added in adherent VERO cell, often organize repetition three holes, be placed in 37 DEG C, 5% CO
2cultivate in incubator.
(3) cultivate after 48 hours, utilize the cytotoxicity of Alamarblue activity detection kit detection of drugs.Read plate instrument (Envison 2102 Multilabel Reader) with PerkinElmer multiple labeling and read fluorescence signal.
(4) each group data calculate using not drug containing matched group as criterion group, computing formula=medicine group/do not add medicine * 100.Result goes out meansigma methods and standard deviation by GraphPad Prism 5 computed in software.
Experimental result display fructose 1,6-diphosphate sodium salt is do not have virose at 1000 μ g/mL to VERO cell, the results are shown in Table 1.
2.2 fructose 1,6-diphosphate sodium salts kill ADV and EV71 virus activity and detect.
(1) the fructose 1,6-diphosphate sodium salt (final concentration is respectively 1000 μ g/mL, 333 μ g/mL, 111 μ g/mL, 37 μ g/mL, 12.3 μ g/mL, 4.1 μ g/mL, 1.37 μ g/mL) of ADV and EV71-GFP viral stock and 3 times of gradient dilutions was 37 DEG C of preincubates 4 hours.Then by virus and medicament mixed diluted sample 100 times of postoperative infection VERO cells; Matched group be then viral stock and each acute drug respectively 37 DEG C of preincubates 4 hours, mix after each dilution 50 times and infect VERO cell immediately.
(2) virus infected cell removes supernatant after cultivating 1 hour at 37 DEG C, washes twice with PBS, removes the virus and residual medicine of not adsorbing, and is then changed to fresh culture and continues to cultivate.
(3) after cultivating 48 hours at 37 DEG C, detected and quantitative analysis cell pathological changes (cytopathic effect, CPE) or the fluorescence intensity of green fluorescent protein (GFP) by high intension, calculate suppression ratio.Suppression ratio (%)=(viral hole fluorescence intensity-sample well fluorescence intensity)/viral hole fluorescence intensity * 100%.
Experimental result display fructose 1,6-diphosphate sodium salt can not deactivation ADV and EV71.
Embodiment 6:1,6-fructose diphosphate sodium detects the viricidal activity of respiratory syncytial virus.
1. experiment material
1.1 cells, virus
Hep-2 cell is the preservation of this laboratory.
Respiratory syncytial virus (RSV) is presented by Wuhan University.
1.2 reagent
DMEM culture medium and FBS are purchased from GIBCO company; Alamarblue and CellTiter Glo cytoactive detection kit is purchased from Promega company.
1.3 experimental apparatus
Multiple labeling microtiter plate reader (PerkinElmer); High intension cytoanalyze (PerkinElmer); Cell culture incubator is Thermo Products.
2. experimental technique and result
2.1 fructose 1,6-diphosphate sodium salts detect the cytotoxicity of Hep-2 cell.
(1) by Hep-2 cell according to 8 × 10
3individual cells/well is inoculated in 96 porocyte culture plates, uses the DMEM culture medium of the penicillin and streptomycin containing 10%FBS, 100U/mL at 37 DEG C, 5% CO
2humidified incubator in cultivate.
(2) with culture medium by fructose 1,6-diphosphate sodium salt from 20mg/ml, 2 times of gradients are diluted to 9 concentration successively, the culture medium of drug containing are directly added in adherent Hep-2 cell, often organize repetition three holes, be placed in 37 DEG C, 5% CO
2cultivate in incubator.
(3) cultivate after 48 hours, utilize the cytotoxicity of Alamarblue activity detection kit detection of drugs.Read plate instrument (Envison 2102 Multilabel Reader) with PerkinElmer multiple labeling and read fluorescence signal.
(4) each group data calculate using not drug containing matched group as criterion group, computing formula=medicine group/do not add medicine * 100.Result goes out meansigma methods and standard deviation by GraphPad Prism 5 computed in software.
Result display fructose 1,6-diphosphate 1000 μ g/mL to Hep-2 cell be do not have virose.
2.2 fructose 1,6-diphosphate sodium salts kill respiratory syncytial virus (RSV) Activity determination.
(1) RSV viral stock and 3 times of gradient dilutions 1,6-fructose diphosphate sodium (final concentration is respectively 1000 μ g/mL, 333 μ g/mL, 111 μ g/mL, 37 μ g/mL, 12.3 μ g/mL, 4.1 μ g/mL, 1.37 μ g/mL) was 37 DEG C of preincubates 3 hours, then (virus infection titer is 100TCID by virus and medicament mixed diluted sample 100 times of postoperative infection Hep-2 cells
50); Matched group be then viral stock and each acute drug respectively 37 DEG C of preincubates 3 hours, mix after each dilution 50 times and infect Hep-2 cell immediately.
(2) virus infected cell removes supernatant after cultivating 1 hour at 33 DEG C, and cell PBS washes twice, removes the virus and residual medicine of not adsorbing, and is then changed to fresh culture and continues to cultivate.
(3) 33 DEG C are continued cultivation after 96 hours, detect cytoactive by CellTiter-Glo, calculate cytoprotective rate, and then learn the suppression ratio of fructose 1,6-diphosphate sodium salt to RSV viricidal activity.Suppression ratio (%)=(sample well-viral hole)/(cell hole-viral hole) * 100%.
Experimental result display fructose 1,6-diphosphate sodium salt energy dose-dependant ground deactivation respiratory syncytial virus, the IC of fructose 1,6-diphosphate Sha Xi road syncytial virus
50be 105 μ g/mL, the results are shown in Table 1.
Table 1 fructose 1,6-diphosphate sodium salt is to enveloped virus broad-spectrum killing virus Activity determination
IC
50: suppression ratio is the drug level of 50%
CC
50: cytotoxicity is the drug level of 50%
The influenza activity that kills of embodiment 7 fructose 1,6-diphosphate sodium salt temperature-independent detects.
1. experiment material
1.1 cells, virus
Madin-Darby canine kidney(cell line) (MDCK) MDCK(ATCC CCL-34) preserved by this laboratory.Influenza A H1N1 hypotype (A/PuertoRico/8/1934) is preserved for this laboratory, is increased and obtain by Embryo Gallus domesticus.Instar chicken embryo was purchased from Beijing Cimmeria Wei Tong laboratory animal Technology Co., Ltd. on 10th.
1.2 reagent
DMEM culture medium is purchased from GIBCO company; Neuraminidase substrate MUNANA is purchased from Sigma-Aldrich.Fructose 1,6-diphosphate sodium salt is purchased from Aladdin.
1.3 experimental apparatus
Multiple labeling microtiter plate reader (PerkinElmer) is Bole's Products; Cell culture incubator is Thermo Products; Electric heating constant temperature tank is purchased from the permanent Science and Technology Ltd. in Shanghai one.
2. experimental technique and result
(1) Influenza virus H1N1 hypotype (A/PuertoRico/8/1934) (6.5 × 10
5tCID
50/ ml) with 1 of 3 times of gradient dilutions, 6-fructose diphosphate sodium (final concentration is respectively 1000 μ g/mL, 333 μ g/mL, 111 μ g/mL, 37 μ g/mL, 12.3 μ g/mL) hatches 4 hours at 4 DEG C, 25 DEG C, 37 DEG C, 42 DEG C respectively, then by virus mixture diluted 100 times of postoperative infection mdck cells with medicine.
(2) virus infected cell removed supernatant after 1 hour, washed the virus and residual medicine removing for twice and do not adsorb with PBS, was then changed to fresh culture and continued to cultivate at 37 DEG C.
After (3) 24 hours, collect supernatant and measure NA(neuraminidase) active, calculate suppression ratio.
As shown in Figure 2, the effect of killing the virus of fructose 1,6-diphosphate sodium salt infected by influenza is temperature-independent to experimental result.When 4 DEG C, fructose 1,6-diphosphate infected by influenza does not kill the virus effect.When temperature is 25 DEG C, fructose 1,6-diphosphate shows the more weak effect of killing the virus.But when 37 DEG C and 42 DEG C, the effect of killing the virus of fructose 1,6-diphosphate significantly improves.
Those skilled in the art will readily understand; the foregoing is only preferred embodiment of the present invention; not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (3)
1.1,6-fructose diphosphate and the application of salt in the medicine and virus inactivating agent of preparation control mankind infection of coated virus formed thereof.
2. apply as claimed in claim 1, it is characterized in that: described mankind's enveloped virus is Japanese B encephalitis virus (JEV), dengue virus (DENV), vesicular stomatitis virus (VSV), respiratory syncytial virus (RSV) or influenza virus.
3. apply as claimed in claim 1, it is characterized in that: described medicine be the salt that formed using fructose 1,6-diphosphate or its as active constituents of medicine, make the pharmaceutically acceptable dosage form of any one.
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| CN1265397A (en) * | 2000-01-17 | 2000-09-06 | 欧阳平凯 | Preparation of magnesium fructose-1,6-diphosphate |
| CN101904856A (en) * | 2010-08-06 | 2010-12-08 | 南京南农高科兽药研究所有限公司 | Application of 1,6-diphosphofructose and derivative thereof in preparing animal medical health care products |
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| CN1265397A (en) * | 2000-01-17 | 2000-09-06 | 欧阳平凯 | Preparation of magnesium fructose-1,6-diphosphate |
| CN101904856A (en) * | 2010-08-06 | 2010-12-08 | 南京南农高科兽药研究所有限公司 | Application of 1,6-diphosphofructose and derivative thereof in preparing animal medical health care products |
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| 丁书文主持: "疑难杂症笔会.病毒性心肌炎", 《山东中医杂志》 * |
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